In recent years, advantages for sol-gel immobilization of biomaterials such as proteins and enzymes have been studied. Nano-structured complexes composed of sol-gel provide a liquid environment to the biomaterials to increase stability and retain biological activity. A sol-gel matrix restricts activity of biomolecules entrapped therein and thus prevents irreversible structural deformation, and this entrapment weakens molecular interactions between the sol-gel matrix and molecular residues exposed to the sol-gel matrix.
The present inventors developed a method for screening an appropriate sol-gel composition appropriate depending on molecular weights and/or diameters of several target molecules immobilized (Kim, S. et al., Analytical chemistry, 78: 7392, 2006), and confirmed sol-gel characterization including adhesive strength, spot shape, transparency, destruction, and entrapping, and successfully immobilized target materials including aptamers in the sol-gel network.
Sol-gel is widely used to entrap macromolecules, but there have been no reports yet that the small molecule compound was successfully entrapped and then allowed to bind to affinity ligand. A sol-gel composition is designed in order to entrap small molecules, but this composition is for attaching sol-gel to the existing substrate such as a micro-titer plate polymer.
Meanwhile, porous silicon (PS) can be produced by anodic etching of monocrystalline silicon and offers a wide range of porous compositions based on the electrochemical process conditions. In the related arts where the porous silicon is used for protein immobilization, enzymes are covalently immobilized thereon (Ressine, A. et al., Nanobiotechnol 4:18, 2008; Ressine, A. et al., Biotechnol Annu Rev 13:149, 2007; Drott, J. et al., Thin Solid Films 330:161, 1998; Drott, J. et al., MikroChimica Acta 131:115, 1999), or antibodies are adsorbed onto a surface thereof (Ressine, A. et al., Analytical chemistry, 75:6968, 2003; Finnskog, D. et al., J Proteome Res 3: 988, 2004). However, the porous silicon compositions described in the related art documents is not appropriate for sol-gel based anchoring and aptamer selection.
Meanwhile, aptamers are probes recognizing specific molecules. Aptamers have high affinities to respective molecules and have extensive targets up to proteins, peptides, composite molecules of drugs, organic small molecules, and metal ions. It has been generally known that aptamers have higher affinity than antibodies.
Typically, aptamers are fabricated through repetition of in vitro selection or systematic evolution of ligand exponential enrichment (SELEX). In order to adapt SELEX to an appropriate tool for selecting high affinity aptamers targeting small molecule compounds, nitrocellulose membrane elution or affinity columns cannot be employed.
Accordingly, the present inventors endeavored to find substrates suitable to spot the sol-gel composition entrapping small molecules thereon, in order to solve the problem in that immobilization of small molecules is not easy in the sol-gel chip of the related art. As a result, the present inventors confirmed that, in the case where a sol-gel composition suitable to entrap small molecules is spotted on a porous silicon surface, an entrapped small molecule compound is well maintained in the spot and easily binds to a small molecule-specific aptamer, and completed the present invention.